analysis of new type iii effectors from xanthomonas uncovers xopb and xops as suppressors of plant...

Analysis of new type III effectors from Xanthomonas uncoversXopB and XopS as suppressors of plant immunity

Sebastian Schulze1, Sabine Kay1, Daniela Buttner1, Monique Egler1, Lennart Eschen-Lippold2, Gerd Hause3,

Antje Kruger1, Justin Lee2, Oliver Muller1, Dierk Scheel2, Robert Szczesny1, Frank Thieme1 and Ulla Bonas1

1Institute of Biology, Department of Genetics, Martin-Luther-University Halle-Wittenberg, Weinbergweg 10, D-06120 Halle (Saale), Germany; 2Leibniz Institute of Plant Biochemistry,

Weinberg 3, D-06120 Halle (Saale), Germany; 3Biozentrum, Martin-Luther-University Halle-Wittenberg, Weinbergweg 22, D-06120 Halle (Saale), Germany

Author for correspondence:Ulla Bonas

Tel: +49 345 5526290Email: [email protected]

Received: 28 March 2012

Accepted: 15 May 2012

New Phytologist (2012)doi: 10.1111/j.1469-8137.2012.04210.x

Key words: bacterial spot disease, Capsicum

annuum (pepper), cell death suppression,effector, HpaB, type III secretion, vesicletrafficking, Xanthomonas campestris.

Summary

• The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas

campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into

plant cells by a type III secretion system and interfere with cellular processes to the benefit of

the pathogen.

• In this study, we analyzed eight T3Es from Xcv strain 85-10, six of which were newly identi-

fied effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS

contribute to disease symptoms and bacterial growth, and suppress pathogen-associated

molecular pattern (PAMP)-triggered plant defense gene expression.

• In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing

defense responses related to both PAMP-triggered immunity (PTI) and effector-triggered

immunity (ETI).

• XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with

eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in

the suppression of ETI-related responses, but still interfered with vesicle trafficking and was

only slightly affected with regard to the suppression of defense gene induction. This suggests

that XopB-mediated suppression of PTI and ETI is dependent on different mechanisms that

can be functionally separated.

Introduction

Plants defend themselves against microbial invaders by basaldefense responses including the production of reactive oxygenspecies, activation of mitogen-activated protein kinase (MAPK)cascades, expression of pathogenesis-related (PR) genes andcallose deposition into the plant cell wall (Nurnberger et al.,2004). Usually, these defense reactions are activated on recogni-tion of pathogen-associated molecular patterns (PAMPs), such asflagellin, lipopolysaccharides and elongation factor EF-Tu, byspecific receptors in the plant plasma membrane (Nurnbergeret al., 2004; Jones & Dangl, 2006). However, specialized bacte-rial pathogens have evolved sophisticated strategies to avoid ormanipulate plant defense responses and to proliferate in theplant’s apoplast. Essential for the pathogenicity of Gram-negativebacteria is often a type III secretion (T3S) system consisting of acomplex membrane-spanning injection apparatus that is associ-ated with an extracellular pilus and a channel-like translocon inthe plant plasma membrane (Buttner & He, 2009). T3S systemstranslocate type III effectors (T3Es) directly into the hostcell cytosol where they interfere with plant cell processes to thebenefit of the pathogen, often leading to the suppression of

PAMP-triggered immunity (PTI) (Jones & Dangl, 2006). How-ever, individual effectors can also act as avirulence (Avr) proteinsthat are recognized in plants carrying a corresponding resistance(R) gene. Recognition leads to the elicitation of host defense reac-tions that often culminate in the hypersensitive response (HR), arapid, localized programmed cell death reaction that restrictspathogen ingress (Klement & Goodman, 1967; Greenberg &Yao, 2004). To circumvent effector-triggered immunity (ETI),bacterial pathogens have evolved T3Es that interfere with theinduction of R gene-mediated defense responses (Jones & Dangl,2006).

Most phytopathogenic bacteria translocate 20–30 differentT3Es into the plant cell (Buttner & Bonas, 2010; Hann et al.,2010). Notably, the deletion of individual effector genes oftendoes not lead to reduced virulence, presumably because of func-tional redundancies among T3Es (Buttner & Bonas, 2010; Hannet al., 2010). Although the molecular functions of most T3Esinside the plant cell are still unknown, a number of T3Es fromphytopathogenic bacteria have been shown to interfere withsignaling cascades, proteasome-dependent protein degradationand the transcription machinery (Kay & Bonas, 2009; Block &Alfano, 2011).

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Our laboratory studies the T3S system and T3Es fromXanthomonas campestris pv. vesicatoria (Xcv, also termedX. euvesicatoria (Jones et al., 2004) and X. axonopodis pv.vesicatoria (Vauterin et al., 2000)), the causal agent of bacterialspot disease on pepper and tomato. The T3S system of Xcv isencoded by the 23-kb chromosomal hrp (hypersensitive responseand pathogenicity) gene cluster, which is essential for bacterialgrowth and disease symptoms on susceptible plants, as well as forHR induction in resistant host and nonhost plants (Bonas et al.,1991). The expression of hrp genes is induced in planta by theOmpR-family regulator HrpG, which controls the expression ofa genome-wide regulon (Noel et al., 2001) including hrpX, whichencodes an AraC-type transcriptional activator (Wengelnik &Bonas, 1996; Wengelnik et al., 1996a). HrpX binds tocis-regulatory PIP (plant-inducible promoter) boxes in thepromoter regions of hrp and other genes that contribute tovirulence (Koebnik et al., 2006).

In addition to a functional T3S apparatus, efficient transloca-tion of effectors by the Xcv T3S system requires the T3S chaper-one HpaB, which has a broad substrate specificity (Buttner et al.,2004, 2006; Szczesny et al., 2010a). T3S chaperones specificallybind T3S substrates and promote their secretion and ⁄ or stability(Parsot et al., 2003; Wilharm et al., 2007). Interestingly, T3Es inXcv differ in their HpaB dependence and are therefore groupedinto two classes. While class A effectors depend on HpaB fortranslocation, class B effectors are translocated even in theabsence of HpaB, albeit in reduced amounts. It is conceivablethat HpaB imposes a hierarchy on T3E translocation and thatclass A effectors are preferentially translocated during a certainstage of the infection process (Buttner et al., 2006).

On the basis of experimental and bioinformatic analyses, indi-vidual Xanthomonas strains express 23–37 different T3Es(Buttner & Bonas, 2010). The largest effector class, although notpresent in all strains, is the AvrBs3 ⁄ PthA family of transcriptionactivator-like (TAL) effectors, which mimic eukaryotic transcrip-tion factors and induce the transcription of plant genes tosupport bacterial growth and dispersal (Marois et al., 2002; Yanget al., 2006; Kay et al., 2007; Boch et al., 2009; Antony et al.,2010). Intriguingly, they can also activate R gene promoters lead-ing to the induction of the HR (Gu et al., 2005; Romer et al.,2007). Plant transcript levels are also modulated by XopD (Xop,Xanthomonas outer protein) from Xcv, which negatively regulatesthe expression of defense- and senescence-related genes via ethyl-ene response factor amphiphilic repression motifs (Kim et al.,2008). In addition, XopD acts as a cysteine protease (Hotsonet al., 2003). All biochemical activities of XopD contribute to itsvirulence function, that is, a delay of plant chlorosis and necrosisand the promotion of bacterial multiplication in tomato (Kimet al., 2008). Cysteine protease activity has also been demon-strated for effectors of the YopJ ⁄ AvrRxv family from plant- andanimal-pathogenic bacteria (Orth et al., 2000; Hotson &Mudgett, 2004; Ma et al., 2006; Sweet et al., 2007; Szczesnyet al., 2010a). Their exact mode of action, however, is a contro-versial issue because an acetyltransferase activity has also beendescribed (Mukherjee et al., 2006). Members of the YopJ ⁄AvrRxv family, for example XopJ and AvrBsT from Xcv, are

involved in the suppression of plant defense by the inhibition ofcell wall-associated defense responses and ETI, respectively(Bartetzko et al., 2009; Szczesny et al., 2010a). A role in thesuppression of plant immunity has also been proposed for otherXcv T3Es, including XopX and XopN (Metz et al., 2005; Kimet al., 2009). The latter presumably suppresses PTI by targetingan atypical receptor-like kinase in tomato involved in immunesignaling (Kim et al., 2009).

Our goal was the identification and analysis of new T3Es fromthe Xcv model strain 85-10, for which 16 T3Es have been identi-fied and confirmed experimentally to date (Mudgett et al., 2000;Escolar et al., 2001; Noel et al., 2002, 2003; Hotson et al.,2003; Roden et al., 2004; Metz et al., 2005; Morales et al.,2005; Lorenz et al., 2008; Szczesny et al., 2010a; Potnis et al.,2011). Analysis of the genome sequence (Thieme et al., 2005)revealed the presence of further effector candidates as a result ofPIP boxes in the corresponding promoter, homology to knowneffectors and sequence motifs typically restricted to eukaryoticproteins. In this study, we analyzed eight T3Es, six of which werenewly identified, using AvrBs3 as T3S reporter. Mutant and over-expression studies revealed that XopB and XopS contribute todisease symptoms and bacterial growth, and suppress plantdefense gene expression. In addition, XopB inhibits cell deathreactions triggered by different T3Es, thus suppressing both PTI-and ETI-related responses.

Materials and Methods

Bacterial strains and growth conditions

Escherichia coli cells were cultivated in LB (lysogeny broth)medium at 37�C. Agrobacterium tumefaciens was grown at 30�Cin YEB (yeast extract broth) medium and Xcv at 30�C in NYG(nutrient yeast glycerol; Daniels et al., 1984), hrp gene-inducingmedium (XVM2; Wengelnik et al., 1996b) or secretion medium(minimal medium A; Ausubel et al., 1996) supplemented with10 mM sucrose and 0.3% casamino acids. Plasmids were intro-duced into E. coli and A. tumefaciens by electroporation, and intoXcv by conjugation, using pRK2013 as helper plasmid intriparental matings (Figurski & Helinski, 1979). Bacterial strainsand plasmids are listed in Supporting Information Table S1.

Plant material and inoculations

The near-isogenic pepper (Capsicum annuum) cultivars ECW,ECW-10R, ECW-20R and ECW-30R (Minsavage et al., 1990),Nicotiana benthamiana and N. tabacum plants were grown at25�C with 65% relative humidity and 16 h light. Xcv strains werehand-inoculated with a needleless syringe into the apoplast ofleaves at 108 colony-forming units (cfu) ml)1 in 10 mM MgCl2.For in planta growth curves, bacteria were inoculated at 104 cfuml)1, and bacterial growth was determined as described by Bonaset al. (1991). For in planta transient expression studies,A. tumefaciens strain GV3101 was grown overnight in YEBmedium, resuspended in inoculation medium (10 mM MgCl2, 5mM MES, pH 5.3, 150 lM acetosyringone) and inoculated into

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leaves at 4 · 108 cfu ml)1 for localization studies and 2 · 108

cfu ml)1 for secGFP secretion assays. For the analysis of celldeath suppression, we used final bacterial densities of 4 · 108 cfuml)1 (Agrobacterium delivering avrBs1, avrBs2, avrBsT, avrRxv,xopG and xopJ) and 6 · 108 cfu ml)1 (Agrobacterium deliveringxopS, xopB and mutant derivatives), respectively. For fraction-ation studies, Agrobacterium strains were inoculated at 6 · 108

cfu ml)1. For co-expression, Agrobacterium strains were mixedbefore inoculation.

RNA analysis

RNA extraction from Xanthomonas, cDNA synthesis and semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) experiments were performed as described by Noelet al. (2001) and Thieme et al. (2007). For oligonucleotidesequences, see Table S2. Experiments were performed at leastthree times for each gene with two independent cDNA prepara-tions each.

Protein analysis

Xanthomonas protein secretion experiments were performed asdescribed by Rossier et al. (1999) and Buttner et al. (2002).Equal amounts of total bacterial cell extracts and culture super-natants were analyzed by sodium dodecylsulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and immunoblotting followingstandard protocols. To check for bacterial lysis, blots wereroutinely reacted with an antibody specific for the intracellularprotein HrcJ (data not shown; Rossier et al., 2000). ForAgrobacterium-mediated expression studies, two 0.64-cm2 leafdisks per strain were frozen in liquid nitrogen, ground and resus-pended in 100 ll of 8 M urea and 50 ll of 5 · Laemmli buffer,and boiled for 10 min. Proteins were separated by SDS-PAGEand analyzed by immunoblotting. We used specific polyclonalantibodies for the detection of AvrBs3 (Knoop et al., 1991), GFP(green fluorescent protein; Invitrogen GmbH, Karlsruhe,Germany), RFP (red fluorescent protein; Antibodies-onlineGmbH, Aachen, Germany), cFBPase (Agrisera AB, Vaennaes,Sweden) and XopB (H. Berndt & U. Bonas, unpublished), and amonoclonal c-Myc-specific antibody (Roche Diagnostics, Mannheim,Germany). Horseradish peroxidase-labeled a-rabbit and a-mouseantibodies (Amersham Pharmacia Biotech, Piscataway, NJ, USA)were used as secondary antibodies. Antibody reactions were visu-alized by enhanced chemiluminescence (Amersham PharmaciaBiotech).

Golden Gate vectors

The suicide vector pOGG2 was derived from the suicide plasmidpOK1 (Huguet et al., 1998) and contains a lacZ gene forblue–white selection. The binary vector pGGA1 contains thebackbone of pBGWFS7 (Karimi et al., 2005), a chloramphenicolresistance-ccdB selection cassette, and allows the expression ofgenes 3¢ translationally fused to GFP under the control of the35S promoter. To allow cloning of DNA fragments by

BsaI ⁄ T4-ligase cut-ligation (Engler et al., 2008), additional BsaIrestriction sites were removed during vector construction. Clon-ing details are available on request.

GATEWAY and Golden Gate expression constructs

To generate binary expression constructs, the coding sequencesof avrRxv, xopB, xopBA313V, xopBK455R, xopG, xopI, xopK, xopM,xopR, xopS and xopV were amplified by PCR, cloned intopENTR ⁄ D-TOPO (Invitrogen) and recombined into pGWB5,pGWB17, pK7FWG2 and pK7WGF2 (Karimi et al., 2005;Nakagawa et al., 2007) using Gateway� technology (Invitro-gen). Oligonucleotides are listed in Table S2.

For expression in Xcv, xopS was amplified from strain 85-10and cloned into the Golden Gate-compatible expression vectorpBRM (Szczesny et al., 2010b). xopB and xopBA313V PCR ampli-cons were cloned downstream of plac into the Golden Gate-compatible expression vector pLAND, which allows the insertionof the cloned fragment into the genome by homologous recombi-nation (C. Lorenz & D. Buttner, unpublished).

To generate avrBs3D2 fusions, the promoters and 5¢ codingsequences of xopG, xopI, xopK, xopM, xopR, xopS and xopV wereamplified by PCR from genomic DNA of Xcv 85-10, cloned intopENTR ⁄ D-TOPO and recombined into pL6GW356 (Noelet al., 2003). In the case of xopM, the complete coding regionwas amplified. For xopB, the 5¢ coding sequence without pro-moter was amplified and cloned downstream of plac intopBR356, which allows a 3¢ fusion of the gene to avrBs3D2 encod-ing an N-terminally truncated AvrBs3 derivative with a C-terminalFLAG epitope (C. Lorenz & D. Buttner, unpublished).

For Arabidopsis protoplast assays, xopB, xopBA313V and xopSwere cloned into pUGW14 (Nakagawa et al., 2007) using thepENTR ⁄ D-TOPO constructs described above. H2B (At5g59910)was recombined into pUGW15 (Nakagawa et al., 2007) using anavailable pDONR221 derivative (Feilner et al., 2005).

secGFP, which contains a basic chitinase signal sequence at theN-terminus of GFP (Haseloff et al., 1997), was generated by clon-ing annealed oligonucleotides into pGGA1. xopJ was recombinedinto pGWB17 (Nakagawa et al., 2007) using an availablepENTR ⁄ D-TOPO construct (Thieme et al., 2007). xopJC235A

was derived from xopJ by splicing by overlap extension(SOE)-PCR, cloned into pENTR ⁄ D-TOPO and recombinedinto pGWB17 (Nakagawa et al., 2007). AtSYP121_Sp2 (Tyrrellet al., 2007) was amplified from Arabidopsis thaliana (Col-0)cDNA, cloned into pENTR ⁄ D-TOPO and recombined intopGWB17 (Nakagawa et al., 2007). Oligonucleotides are listed inTable S2.

Effector deletion strains

To generate deletions of xopG, xopI, xopM, xopR, xopS and xopV,0.6–1-kb fragments upstream and downstream of the respectivegene were amplified from genomic DNA of Xcv 85-10 by PCRusing oligonucleotides harboring appropriate restriction sites(Table S2). For the deletion of xopK, corresponding fragmentswere synthesized by Eurofins MWG Operon (Ebersberg,

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Germany). Fragments were cloned into the suicide vectorspK18mobsac (Schafer et al., 1994) (xopG, xopI, xopM), pOK1(Huguet et al., 1998) (xopS) or pOGG2 (xopK, xopR, xopV). Theresulting constructs were conjugated into Xcv strain 85-10, andmutants were selected by PCR. The xopB deletion mutant wasavailable (Noel et al., 2001).

Mesophyll protoplast transient expression assay

Transient expression experiments with A. thaliana (Col-0)-derived protoplasts were performed according to Ranf et al.(2011). Protoplast samples were co-transformed with the NHL10promoter-luciferase construct (Boudsocq et al., 2010; Ranf et al.,2011), pUBQ10-GUS (Norris et al., 1993) and eitherp35S-effector gene constructs (xopB, xopBA313V, xopS) orp35S-H2B (At5g59910) as a control (10 lg total DNA per 100ll protoplasts; ratio 1 : 1 : 1).

Electrolyte leakage measurements

Triplicates of five leaf disks each (0.64 cm2) were harvested at 1and 2 d post-inoculation (dpi), respectively. Measurements werecarried out as described by Szczesny et al. (2010a).

Microscopy

Lower epidermal cells of N. benthamiana were inspected with aconfocal laser scanning microscope LSM 510 and LSM ImageBrowser software (Carl Zeiss GmbH, Gottingen, Germany)according to the manufacturer’s protocol. mCherry Golgi (G-rk,CD3-967) was used for co-localization experiments (Nelsonet al., 2007). To visualize plant cell nuclei, leaves were infiltratedwith 0.1% (w ⁄ v) 4’,6-diamidino-2-phenylindole (DAPI)

solution 1 h before inspection. GFP was excited with an argonlaser at 488 nm, mCherry with an HeNe laser at 543 nm andDAPI with a krypton (UV) laser at 364 nm. The emission filterwavelengths were 505–530 nm for GFP, 560–615 nm formCherry and 385–470 nm for DAPI.

Transmission electron microscopy was performed as describedby Thieme et al. (2007) using an EM Libra 120 (Carl ZeissGmbH).

Results

Gene regulation of xopB, xopG and six new T3E genecandidates in Xcv strain 85-10

For the analysis of effector proteins from Xcv strain 85-10, wechose XopB, previously shown to be type III secreted into themedium (Noel et al., 2001), and XopG, a recently identifiedT3E (Potnis et al., 2011) with homology to the HopH1 familyfrom Pseudomonas syringae (Thieme et al., 2005). In addition, weidentified six new candidate effectors in Xcv strain 85-10 as aresult of homology to known effectors (XopK, XopR and XopV),predicted eukaryotic motifs (XopI), indication of gene acquisi-tion by horizontal gene transfer because of significantly lower G+ C content (xopS) and the presence of a PIP box in the respec-tive promoters (xopI, xopM, xopR and xopS) (Table 1). Becausethe presence of a PIP box suggests the co-regulation of a genewith the T3S system, we performed RT-PCR analyses of xopGand the candidate effector genes in the Xcv wild-type strain85-10, its derivative 85*, which expresses a constitutively activeHrpG point mutant resulting in constitutive expression of theT3S system (Wengelnik et al., 1999), and the hrpX deletionmutant 85*DhrpX (Noel et al., 2001). When the bacteria werecultivated in complex NYG medium, mRNA of xopG was

Table 1 Characteristics of effector genes from Xanthomonas campestris pv. vesicatoria (Xcv) strain 85-10 analyzed in this study

Gene (gene no.) G + C (%)a Comment(s)b

Homolog inc

PIP boxd Co-reg.e Tr. cl.fX. P. R. A. o.

xopB (XCV0581) 55.54 HopD family (Lindeberg et al., 2005) + + + + +g + + BxopG (XCV1298) 52.02 Putative zinc metalloprotease, HopH family (Lindeberg et al., 2005) + + + + ) ) ) BxopI (XCV0806) 65.11 F-box motif + ) ) ) ) + + BxopK (XCV3215) 66.60 Homology to XOO1669 (Furutani et al., 2009) + ) ) + +h ) + BxopM (XCV0442) 62.76 No homology to known effectors + + ) + +i + + BxopR (XCV0285) 66.34 Homology to XOO4134 (Furutani et al., 2009) + ) ) ) ) + + AxopS (XCV0324) 55.34 No homology to known effectors + ) ) ) ) + + AxopV (XCV0657) 61.50 Homology to XOO3803 (Furutani et al., 2009) + ) + + ) ) + B

aG + C content of the DNA within the coding region (G + C content of the Xcv 85-10 chromosome: 64.75% (Thieme et al., 2005)).bPutative function of gene product, eukaryotic motifs and homology to known type III effectors.cHomologs were determined using BLAST algorithms. ), absence; + presence of a (partial) homolog. X., Xanthomonas spp.; P., Pseudomonas spp.; R.,Ralstonia solanacearum; A., Acidovorax spp.; o., other organisms.dPresence of a PIP and -10 box (TTCGB-N15-TTCGB-N30–32-YANNNT; B represents C, G, or T; Y represents C or T) in the respective promoter (Koebniket al., 2006). +, presence; ), absence of distinct motifs.eHrpG- and HrpX-dependent co-regulation with the T3S system (+, co-regulation; ), constitutive expression).fTranslocation class; classification based on HpaB dependence (Buttner et al., 2006).gPantoea agglomerans pv. gypsophilae.hBurkholderia rhizoxinica.iCollimonas fungivorans.

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detectable at similar levels in strains 85-10, 85* and 85*DhrpX,suggesting constitutive expression (Fig. 1a). The transcripts ofxopB, xopI, xopK, xopM, xopR, xopS and xopV were amplifiedfrom strain 85*, suggesting co-expression with T3S genes. As theamounts of amplified transcripts were clearly reduced with RNApreparations from strains 85-10 and 85*DhrpX, transcription ofthe candidate genes is presumably controlled by both HrpG andHrpX. The HrpX-dependent induction of xopR has beendescribed previously (Koebnik et al., 2006).

Secretion and translocation of the T3Es

To investigate whether the effector candidates are indeed type IIIdependently secreted and translocated into the plant cell, we

generated translational fusions with the reporter proteinAvrBs3D2, a derivative of the TAL effector AvrBs3 which lacks aT3S and translocation signal (Szurek et al., 2002; Noel et al.,2003). Fusion of a functional T3S signal to AvrBs3D2 enablesits translocation and thus the induction of the HR in pepper cul-tivar ECW-30R plants that harbor the corresponding resistancegene Bs3 (Noel et al., 2003; Thieme et al., 2007). The nativepromoters and 5¢ coding regions of candidate genes (xopG, xopI,xopK, xopR, xopS and xopV) or the complete coding region(xopM) were fused to avrBs3D2. In the case of xopB, the 5¢ codingregion without promoter was used and the fusion construct wasexpressed from the lac promoter. As controls, we used an emptyvector and avrBs3D2 alone (Szurek et al., 2002). All plasmidswere conjugated into Xcv strain 85* and the T3S mutant

(a) (c)

(b)

Fig. 1 Regulation of type III effector (T3E) gene expression and type III-dependent transport of XopB, XopG and six new T3Es. (a) Expression studies ofeffector gene candidates and xopG by reverse transcription-polymerase chain reaction (RT-PCR). Gene-specific fragments were amplified from cDNAderived from Xanthomonas campestris pv. vesicatoria (Xcv) strains 85-10, 85* and 85*DhrpX grown in nutrient yeast glycerol (NYG) medium (Danielset al., 1984). 16S rRNA was amplified as constitutive control. The amplicons were separated on a 1% agarose gel and stained with ethidium bromide. Theexperiment was performed three times with similar results. (b) Type III secretion (T3S) assays of AvrBs3D2 fusion proteins. Strains 85* and 85*DhrcV

ectopically expressing effector (candidate)-AvrBs3D2 fusions were grown in secretion medium. Numbers correspond to the amino acids fused to AvrBs3D2.Equal protein amounts of total cell extracts (TE) and culture supernatants (SN) were analyzed by immunoblotting using an AvrBs3-specific antibody.(c) In planta translocation assay with effector (candidate)-AvrBs3D2 fusions. Xcv strains 85*, 85*DhrcV and 85*DhpaB expressing the effector gene-avrBs3D2 fusions and 85* carrying an empty vector (ev) or expressing avrBs3D2 alone were inoculated into leaves of AvrBs3-responsive ECW-30R pepperplants. Leaves were harvested at 3 d post-inoculation (dpi) and bleached in ethanol for better visualization of the hypersensitive response (HR).






85*DhrcV, which lacks an essential inner membrane componentof the T3S system (Rossier et al., 2000). When the bacteria wereincubated in T3S medium, XopB1–177-, XopG1–100-, XopI1–140-,XopK1–74-, XopM1–520-, XopR1–152-, XopS1–157- andXopV1–148-AvrBs3D2 were detected in the culture supernatantof strain 85*, but not of 85*DhrcV, by an AvrBs3-specific anti-body (Fig. 1b). These results demonstrate that the effector candi-dates contain functional T3S signals in their N-terminal regions.

To test for type III-dependent translocation, Xcv strains 85*and 85*DhrcV expressing avrBs3D2 or the corresponding effectorfusions, as described above, were inoculated into leaves ofAvrBs3-responsive pepper plants (ECW-30R) and the near-isogenic susceptible pepper line ECW, which lacks the Bs3 resist-ance gene. Derivatives of strain 85* expressing XopB1–177-,XopG1–100-, XopI1–140-, XopK1–74-, XopM1–520-, XopR1–152-,XopS1–157- and XopV1–148-AvrBs3D2 induced the HR inECW-30R (Fig. 1c), but not in ECW (data not shown). Asexpected, no HR induction was observed in plants infected withderivatives of strain 85*DhrcV (Fig. 1c). Taken together, thesefindings confirm the type III-dependent secretion and translocationof XopB, XopG, XopI, XopK, XopM, XopR, XopS and XopV,and thus their nature as T3Es. In case of XopG, our data confirm arecent publication which showed type III-dependent translocationof the protein using AvrBs2 as reporter (Potnis et al., 2011).

HpaB-dependent translocation of the T3Es

It has been shown previously that the translocation of some T3Esfrom Xcv is dependent on the general T3S chaperone HpaB(Buttner et al., 2004, 2006). To address this question for the Xcveffectors analyzed here, we introduced the T3E-AvrBs3D2 fusionconstructs into strain 85*DhpaB and inoculated the bacteria intoleaves of resistant ECW-30R pepper plants. As shown inFig. 1(c), XopB1–177-, XopG1–100-, XopI1–140-, XopK1–74-,XopM1–520- and XopV1–148-AvrBs3D2 induced the HR even inthe absence of HpaB, albeit more or less reduced compared withderivatives of strain 85*. By contrast, XopR1–152- andXopS1–157-AvrBs3D2 failed to induce the HR when analyzed instrain 85*DhpaB, although both proteins were expressed (Sup-porting Information, Fig. S1). Thus, according to the publisheddefinition (Buttner et al., 2006), XopR and XopS belong to classA, which includes effectors that are not detectably translocated inthe absence of HpaB, whereas XopB, XopG, XopI, XopK, XopMand XopV, which are still translocated by the 85*DhpaB strain,belong to class B (Table 1).

XopB and XopS contribute to the virulence of Xcv strain85-10

To study the contribution of the T3Es to bacterial virulence, alleffector genes were individually deleted in Xcv strain 85-10, andthe mutants were inoculated into leaves of susceptible ECWpepper plants. In addition, induction of the HR in pepperECW-10R was analyzed, which is based on the recognition of theT3E AvrBs1 by the Bs1 resistance gene (Cook & Stall, 1963;Ronald & Staskawicz, 1988; Escolar et al., 2001). Bacterial strains

carrying deletions of xopG, xopI, xopK, xopM, xopR and xopVshowed no difference in the induction of disease symptoms andthe HR compared with wild-type strain 85-10 (data not shown).By contrast, deletion of xopB or xopS led to significantly reduceddisease symptoms, whereas the HR induction was not impaired(Fig. 2a,b and data not shown). The mutant phenotypes of85-10DxopB and 85-10DxopS were complemented by ectopicexpression of the respective effector gene, suggesting that reducedvirulence was not caused by polar effects of the deletions on down-stream genes (Fig. 2a,b). Although the growth of both individualeffector mutants in ECW plants did not differ significantly fromthat of the wild-type strain (Fig. S2), multiplication of an85-10DxopBDxopS double mutant was reduced significantly, sug-gesting that XopB and XopS fulfill redundant functions (Fig. 2c).

XopB and XopS suppress defense gene expression

To test whether the positive effect of XopB and XopS on diseasesymptoms and bacterial growth can be explained by the suppres-sion of the plant PTI, we analyzed the influence of the effectorson basal and PAMP-induced defense-related gene expression.Therefore, we performed Arabidopsis leaf protoplast assays, awell-established system for PAMP signaling studies (Boudsocqet al., 2010; Ranf et al., 2011). We tested the influence of XopB,a XopB mutant derivative (XopBA313V, see the following section)and XopS on the activity of the A. thaliana NHL10(NDR1 ⁄ HIN1-LIKE 10) (Zipfel et al., 2004) promoter fused tothe firefly luciferase gene (LUC) after application of different elic-itor-active epitopes of bacterial PAMPs. The luciferase reporterassays showed that the expression of xopB and xopS decreased thepNHL10 basal activity significantly, that is, in the absence of anelicitor (Fig. 3a). In addition, both effectors completely inhibitedthe activation of pNHL10 by flg22, a bacterial flagellin epitope(Felix et al., 1999), or elf18, a fragment of bacterial EF-Tu(Kunze et al., 2004) (Fig. 3b,c). XopBA313V was only affectedslightly in its ability to suppress the elf18-dependent pNHL10induction (Fig. 3c). The flg22-mediated induction of pNHL10depends, at least in part, on MAPKs (Boudsocq et al., 2010).Therefore, the activation of the MAPKs MPK3, MPK4, MPK6and MPK11, which are involved in plant immune signaling (Tenaet al., 2011; Bethke et al., 2012), might be affected by XopB andXopS. However, immunoblot analysis using an antibody that spe-cifically detects activated kinases revealed no differences in MAPKactivity between protoplasts expressing the respective effectorgenes and protoplasts expressing CFP (cyan fluorescent protein) asnegative control (Fig. S3; Methods S1). The T3E AvrPto fromP. syringae served as a positive control (He et al., 2006). Takentogether, XopB and XopS suppressed both the basal andPAMP-induced activity of the NHL10 promoter, but they proba-bly act downstream or independent of MAPK activation (Fig. S2).

XopB, XopG, XopM and XopS trigger cell death in differentSolanaceae

To identify additional virulence phenotypes, as well as defensereactions, mediated by the analyzed T3Es, we inoculated leaves

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of pepper ECW, N. benthamiana and N. tabacum, the latter twobeing nonhost plants of Xcv 85-10, with Agrobacterium strainsmediating the in planta expression of the eight effector genesfused to GFP. XopB triggered a cell death reaction inN. benthamiana at 5–6 dpi, but not in N. tabacum (Fig. 4a).These data are in accordance with recent findings (Salomonet al., 2011). We also tested the transient expression of two xopBderivatives with point mutations, accidentally introduced duringPCR amplification, for their cell death-inducing activity.Although XopBK455R was still active, XopBA313V did not elicitcell death in N. benthamiana (Fig. 4b). Immunoblot analysis ofprotein extracts from infected plant material, using a GFP-specific antibody, revealed that XopBA313V protein levels werereduced slightly compared with the wild-type protein. We there-fore inoculated a dilution series of Agrobacterium strains. TheAgrobacterium strain mediating xopB expression triggered celldeath even at low density, corresponding to low XopB proteinamounts in the plant tissue, whereas Agrobacterium-mediatedsynthesis of XopBA313V did not induce any visible cell death reac-tions (Fig. S4). This suggests that functional loss rather than areduced protein level is responsible for the lack of cell deathinduction by XopBA313V.

We also observed a XopG-triggered HR-like cell death inpepper ECW and N. tabacum at 2–3 dpi. Furthermore, XopMelicited a cell death reaction in N. benthamiana at 3–5 dpi, andXopS caused a weak necrosis (compared with the XopG-triggeredreaction) in pepper ECW at 3–4 dpi (Fig. 4a). No distinct plantreactions were observed with the other effectors, although theywere expressed, as confirmed by immunoblot analyses of protein

extracts from N. benthamiana leaves using a GFP-specific anti-body (data not shown).

XopB suppresses cell death reactions triggered by XopGand other T3Es

As described above, XopG induces the HR in pepper ECWwhen transiently expressed in planta, whereas Xcv 85-10, whichnaturally expresses XopG, does not. The latter might be causedby other Xanthomonas T3Es with cell death suppressing (CDS)activity. Therefore, we tested whether XopB or XopS, whichcontribute to bacterial virulence (see earlier in the Resultssection), suppress the XopG-elicited cell death reaction.Co-expression experiments using Agrobacterium-mediated genedelivery revealed that the XopG-dependent HR in pepper andN. tabacum was strongly reduced or fully abolished in the pres-ence of XopB, but not XopS (Fig. 5a). To validate this finding,we used electrolyte leakage assays, a quantitative measure ofearly plant cell death (Stall et al., 1974), and found that XopBcompletely suppressed the XopG HR in N. tabacum at earlytime points (Fig. 5b). Interestingly, no HR suppression wasobserved with XopBA313V, whereas XopBK455R showedwild-type XopB activity (Fig. 5a,b), although the in plantaexpression of both effectors could be detected (Fig. 5c). Hence,the CDS activity in N. tabacum and pepper (Fig. 5a,b) andnecrosis induction in N. benthamiana (Fig. 4b) seem to befunctionally linked.

To explore whether the CDS activity of XopB is restricted toXopG-mediated cell death, we transiently co-expressed xopB with

(a) (b)

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Fig. 2 XopB and XopS contribute to disease symptoms and bacterial growth in planta. (a) Leaves of pepper (Capsicum annuum) cv ECW plants were inoc-ulated with Xanthomonas campestris pv. vesicatoria (Xcv) strains 85-10, 85-10DxopB (DxopB) and an 85-10DxopB derivative in which xopB under controlof the lac promoter was integrated into the genome (+XopB) (see the Materials and Methods section). Photographs were taken at 7 d post-inoculation(dpi). (b) Pepper ECW plants were inoculated with Xcv strains 85-10 and 85-10DxopS (DxopS) carrying the empty vector ()) or pBRM:xopS that expressesxopS from plac (+XopS). Photographs were taken at 7 dpi. (c) Bacterial growth of Xcv strains in leaves of susceptible pepper ECW. The following strainswere inoculated: Xcv 85-10 (wt), 85-10DxopBDxopS (DxopBDxopS) carrying the empty vector (ev) or pBRM:xopS, and the T3S mutant 85-10DhrcN

(DhrcN). Bacterial multiplication was monitored over a period of 9 d. Values represent the mean of three samples from three different plants. Error barsindicate standard deviations. Letters a and b indicate statistically significant differences (t-test, P < 0.01). Experiments were repeated at least three timeswith similar results.






avrBs1, avrBs2, avrBsT, avrRxv and xopJ, and tested for cell deathinduction in corresponding resistant plants. XopB suppressed thecell death reactions elicited by AvrBsT, AvrRxv and XopJ inN. benthamiana (Fig. 6a). The AvrBs1- and AvrBs2-dependentHRs in pepper ECW-10R and ECW-20R, respectively, werenot affected (data not shown). Similar to the effect on

XopG-triggered cell death, XopBA313V exhibited no suppressionactivity, whereas XopBK455R did. Expression of the effector geneswas confirmed by immunoblot (Fig. 6b).

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Fig. 3 XopB and XopS inhibit basal and pathogen-associated molecular pattern (PAMP)-induced defense gene expression. Arabidopsis thaliana Col-0protoplasts were co-transformed with pNHL10-LUC (luciferase) as a reporter, the indicated p35S-effector gene constructs or p35S-H2B (control), andpUBQ10-GUS (b-glucuronidase) for normalization; 14 h after transformation, protoplasts were treated with (a) H2O, (b) 100 nM flg22 and (c) 100 nMelf18, and luciferase activity was monitored for 3 h. Results are depicted as LUC ⁄ GUS ratios. Asterisks indicate statistically significant differences betweenXopB and XopBA313V (Kruskal–Wallis ⁄ Dunn’s post test; *, P < 0.05; **, P < 0.01). The differences between effector-treated and control samples werestatistically significant at every time point (Kruskal–Wallis ⁄ Dunn’s post test; P < 0.001). The experiment was repeated three times with similar results.

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AvrBsT-dependent HR in pepper using Xanthomonas infection.Xcv-mediated additional ectopic expression of xopB, but notxopBA313V, suppressed the HR caused by Xcv strain 75-3, whichnaturally expresses avrBsT, in pepper ECW (Fig. 7a,b). This wascorrelated with increased bacterial growth (Fig. 7c). If avrBsTwas deleted, strain 75-3 caused no HR and grew significantlybetter, as expected. In this case, xopB overexpression provided no

additional growth advantage. This indicates that the positiveeffect of XopB on bacterial growth is based on the specificsuppression of AvrBsT-induced defense responses, and thereforerepresents a biologically relevant activity of the effector.

XopB localizes to Golgi vesicles and the cytoplasm

Analysis of the subcellular localization of T3Es might providesome clues about their site of action inside the plant cell. Toinvestigate the localization of XopB, we transiently expressed axopB::GFP fusion in N. benthamiana using Agrobacterium-mediated gene delivery. Subcellular localization was determinedby confocal laser scanning microscopy at 24 h post-inoculation(hpi). GFP alone was clearly detectable in the cytoplasm andnuclei (Fig. 8a). By contrast, the fluorescence of XopB::GFP wasconfined to vesicle-like structures and the cytoplasm, and was notdetectable in the nucleus (Fig. 8a). We assumed that the vesi-cle-like structures might be part of the Golgi system. To test thishypothesis, we transiently co-expressed XopB::GFP withGolgi-mCherry, a fluorescence marker for the Golgi apparatus(Nelson et al., 2007). Both proteins co-localized (Fig. 8a), sug-gesting that XopB indeed associates with Golgi vesicles. On theultrastructural level, electron microscopy following immunola-beling showed that, in contrast to free GFP, XopB::GFPpredominantly localized to vesicle structures and was stronglyunder-represented in vesicle-free areas (Fig. 8b).

Furthermore, immunoblot of the subcellular fractionation ofN. benthamiana extracts confirmed that XopB::c-Myc, similarto Golgi-mCherry, is predominantly associated with the plantmembrane fraction (Fig. S5; Methods S1). In addition, intactXopBA313V and XopBK455R were detectable (Fig. S6) andshowed a similar localization pattern to the wild-type protein inmicroscopic as well as fractionation studies (Figs 8a and S5).This suggests that the functional loss of XopBA313V is notcaused by mislocalization.

XopB interferes with plant cell protein secretion

The Golgi apparatus provides the cellular basis for intracellularvesicle trafficking, for example protein transport to the plasmamembrane and secretion into the apoplast, which plays an impor-tant role in plant immunity and has been shown to be targetedby several T3Es (Bartetzko et al., 2009; Frey & Robatzek, 2009).To investigate whether XopB interferes with protein secretion ofthe plant cell, we used secGFP, a GFP variant that is secreted tothe apoplast (Batoko et al., 2000). secGFP only accumulates in afluorescent form when its transport to a post-Golgi compartmentis prevented, for example, by the secretion inhibitor Brefeldin A(BFA). To analyze the influence of XopB on secGFP secretion,both proteins were transiently co-expressed in N. benthamianamediated by Agrobacterium. As positive controls, we used BFAand AtSYP121_Sp2, a cytosolic fragment of a plasma mem-brane-bound regulator of vesicle trafficking, which has a domi-nant negative effect on membrane trafficking (Tyrrell et al.,2007), and, in addition, XopJ, a T3E from Xcv which suppressesplant cell secretion (Bartetzko et al., 2009). XopJC235A, a XopJ

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Fig. 4 XopB, XopG, XopM and XopS trigger cell death in differentSolanaceae. (a) Agrobacterium strains mediating the expression of GFP,xopB::GFP, xopG::GFP, xopM::GFP and xopS::GFP, under the control ofthe 35S promoter, were inoculated into leaves of pepper (Capsicum

annuum) cv ECW, Nicotiana benthamiana and N. tabacum. Plantreactions were documented 4 d (ECW) and 6 d (N. benthamiana,N. tabacum) post-inoculation (dpi). Please note that Agrobacterium alonecauses a necrotic reaction in pepper that starts at 3–4 dpi.(b) Agrobacterium strains carrying the empty vector (ev) or binaryconstructs encoding XopB::c-Myc, XopBA313V::c-Myc andXopBK455R::c-Myc, under the control of the 35S promoter, were inoculatedinto N. benthamiana. Photographs were taken at 6 dpi.






mutant that does not block secretion served as a negative control(Bartetzko et al., 2009). Only weak fluorescence was detectableinside the epidermal cells if secGFP was expressed either alone ortogether with the negative control (Fig. 9a). By contrast,co-expression of secGFP and XopB resulted in a strong accumu-lation of GFP fluorescence, forming an intracellular reticulatepattern, comparable with the fluorescence pattern obtained afterco-expression of secGFP with AtSYP121_Sp2 and XopJ or theapplication of BFA (Fig. 9a). XopBA313V also resulted in theaccumulation of intracellular secGFP fluorescence; however, lessdistinct networks and punctate structures were observed(Fig. 9a). By contrast, XopBK455R caused a fluorescence patternindistinguishable from that induced by wild-type XopB.Immunoblot analysis showed that differences in secGFP fluores-cence were not a result of different protein levels (Fig. 9b).

Discussion

In this study, we analyzed eight Xcv effector proteins, six of whichwere newly identified, so that there are now 23 experimentallyverified T3Es in Xcv strain 85-10. A major finding is that XopBis a virulence factor that suppresses plant PTI as well as ETI. TheT3Es were classified on the basis of whether or not their

translocation into plant cells requires the general chaperoneHpaB. XopR and XopS belong to Xcv translocation class A, com-prising T3Es whose translocation into plant cells is completelydependent on HpaB, whereas XopB, XopG, XopI, XopK, XopMand XopV were assigned to class B, because they are still trans-located in the absence of HpaB (Buttner et al., 2006). Both newclass A effectors lack homology to known proteins or motifs, sothat their molecular function remains elusive. By contrast, theclass B effectors comprise the putative enzyme XopG, a memberof the HopH family (Lindeberg et al., 2005) of putative zincmetalloproteases. Other effectors possess interesting features, forexample XopI contains an F-box motif typical for eukaryotic pro-teins playing a role in the ubiquitin-26S proteasome system(UPS). The UPS controls protein stability in eukaryotes(Willems et al., 2004) and appears to be a favorable target formany T3Es, for example members of the GALA family, whichstrongly contribute to the virulence of R. solanacearum (Angotet al., 2006), and the E3 ubiquitin ligase AvrPtoB fromP. syringae (Abramovitch et al., 2006; Janjusevic et al., 2006).

As HpaB is essential for pathogenicity, it was speculated thatclass A effectors play a key role in the establishment of a patho-genic interaction of Xanthomonas with the host (Buttner et al.,2004, 2006). Indeed, XopS is involved in the severity of disease

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Fig. 5 XopB suppresses XopG-dependent cell death in pepper (Capsicum annuum) and tobacco (Nicotiana tabacum). (a) Agrobacterium strains mediatingthe transfer of T-DNAs encoding XopB::c-Myc, XopBA313V::c-Myc, XopBK455R::c-Myc, XopS::c-Myc and XopG::GFP were inoculated into leaves of pepperECW and N. tabacum. For co-expression, the respective strains were mixed before inoculation. Photographs were taken at 4 d post-inoculation (dpi).Pepper leaves were bleached in ethanol for better visualization of cell death reactions. (b) Quantification of XopG-triggered cell death by electrolyte leak-age measurements. The inoculation of N. tabacum was carried out as described in (a). Leaf tissue of infected plants was harvested at 1 dpi (light gray bars)and 2 dpi (dark gray bars). Bars represent the average conductivity for triplicates of five leaf disks; error bars indicate standard deviations. Asterisks indicatestatistically significant differences (t-test, P < 0.01). (c) Co-expression of XopB and XopS with XopG::GFP in N. tabacum. Plants were inoculatedas described in (a), and infected leaf tissue was harvested at 24 hpi and analyzed by immunoblotting using c-Myc- and GFP-specific antibodies. Allexperiments were repeated three times with similar results.

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symptoms, the promotion of bacterial growth and the suppres-sion of PTI, whereas the role of XopR in the virulence of Xcvstrain 85-10 is probably more subtle. Interestingly, deletion ofthe xopR homolog in X. oryzae pv. oryzae resulted in reducedvirulence on host rice plants (Akimoto-Tomiyama et al., 2012).The fact that individual deletion mutants revealed no major con-tribution of XopR and most other effectors to the virulence ofXcv 85-10 is not unexpected and is probably caused by functionalredundancies. Surprisingly, the class B effector XopB clearly con-tributed to the disease symptoms and bacterial growth of 85-10on susceptible plants and the suppression of PTI (Figs 2 and 3).This finding differs from our previous study, where no differencein disease symptoms was observed between the wild-type strainand xopB mutant (Noel et al., 2001), and may be caused by dif-ferent environmental conditions (glasshouse, growth chamber).In addition to its effect on PTI, XopB suppressed the ETI-relatedHR induced by AvrBsT, as well as cell death reactions triggeredby XopG, XopJ and AvrRxv (Figs 5 and 6). Although XopBlocalizes to the Golgi system and the cytoplasm, the inducers ofcell death reactions suppressed by XopB localize to different cel-lular compartments: XopG to the nucleus (Fig. S7), XopJ to theplasma membrane (Thieme et al., 2007), AvrRxv to the cyto-plasm (Bonshtien et al., 2005) and AvrBsT to the nucleus andcytoplasm (Szczesny et al., 2010a). As the co-expression of T3Eswith XopB did not change their subcellular localization (Fig. S8),XopB probably does not interfere with effector recognition, butrather with downstream signaling.

XopJ, AvrBsT and AvrRxv are members of the YopJ ⁄ AvrRxvfamily from plant and animal pathogens and contain a con-served catalytic triad which is essential for cell death induction(R. Szczesny and U. Bonas, unpublished; Orth et al., 2000;Bonshtien et al., 2005; Cunnac et al., 2007). YopJ ⁄ AvrRxvfamily proteins display acetyltransferase and ⁄ or cysteine prote-ase activities (Orth et al., 2000; Ma et al., 2006; Mukherjeeet al., 2006; Sweet et al., 2007; Szczesny et al., 2010a).Although a number of bacterial T3Es possess CDS activity(Jackson et al., 1999; Tsiamis et al., 2000; Abramovitch et al.,2003; Espinosa et al., 2003; Jamir et al., 2004; Lopez-Solanillaet al., 2004; Fujikawa et al., 2006; Fu et al., 2007; Guo et al.,2009; Macho et al., 2010), effectors that have successfully beentested for the suppression of several different cell death pheno-types are the exception. To our knowledge, the only knownexample so far is the sequence-unrelated AvrPtoB fromP. syringae, which inhibits the HR induced by the T3EsAvrPto, HopA1 and AvrRpm1, the fungal avirulence proteinAvr9 and the pro-apoptotic mouse protein Bax (Abramovitchet al., 2003; Guo et al., 2009). Interestingly, AvrPtoB alsoinhibits PTI and promotes bacterial multiplication in planta(Gohre et al., 2008). Suppression of both ETI and PTIdepends on the E3 ubiquitin ligase activity of AvrPtoB(Abramovitch et al., 2006; Janjusevic et al., 2006; Gohre et al.,2008; Gimenez-Ibanez et al., 2009), which targets receptorkinases at the plasma membrane (Gohre et al., 2008;Gimenez-Ibanez et al., 2009).

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Fig. 6 XopB suppresses cell death reactions induced by XopJ, AvrRxv and AvrBsT. (a) Agrobacterium strains mediating the transfer of T-DNA encodingXopB::c-Myc, XopBA313V::c-Myc, XopBK455R::c-Myc, AvrBsT::GFP, AvrRxv::c-Myc and XopJ::GFP were inoculated into Nicotiana benthamiana leaves asindicated. For co-expression, strains were mixed before inoculation. Photographs were taken at 4 d post inoculation (dpi). (b) Expression of the effectorgenes in planta. N. benthamiana leaves were inoculated with Agrobacterium as described in (a). Infected tissue was harvested at 30 h post-inoculation(hpi) (AvrBsT::GFP) and 40 hpi (AvrRxv::c-Myc and XopJ::GFP), and analyzed by immunoblotting using c-Myc- and GFP-specific antibodies. Theexperiments were repeated at least twice with similar results.






In contrast with AvrPtoB, it is unlikely that XopB acts on topof signaling cascades because it does not inhibit the flg22-triggered activation of MAPKs (Fig. S3) and the CDS activity ofXopB is not dependent on membrane-bound receptor kinasesbecause XopG, AvrRxv and AvrBsT are probably not recognizedat the plasma membrane (see earlier in the Discussion section).XopB may therefore target a later step of the convergent cellularpathways following T3E recognition. Our studies point toXopB-dependent inhibition of intracellular vesicle trafficking as apossible mode of action to suppress plant immunity. Vesicle traf-ficking plays an important role in plant defense, for example forthe correct localization of PAMP receptors in the plasma mem-brane. During PTI, genes encoding receptor kinases are induced,including the PAMP receptors themselves (Zipfel et al., 2006;Miya et al., 2007). This results in an increase in receptors and anamplification of the PAMP response (Zipfel et al., 2006). Inaddition, vesicle transport is involved in the export of antimicro-bial molecules, for example PR proteins, phytoalexins and cellwall-bound compounds, and the localization of plasma mem-brane ABC transporters, which release small antimicrobial mole-cules to the cell surface (Kwon et al., 2008). Intriguingly, ourstudies suggest that the inhibition of vesicle transport mightexplain the XopB effect on PTI, but is insufficient for ETI

suppression. XopBA313V completely loses CDS activity, althoughit still inhibits secGFP secretion and is only slightly affected inPTI suppression (Fig. 3). This suggests that the suppression ofPTI and ETI is mediated by separate activities of XopB, and thatETI suppression involves as yet unknown mechanisms.

There are two other T3Es from phytopathogenic bacteriawhich suppress immunity and interfere with plant proteinsecretion: (1) HopM1 from P. syringae, which is targeted toArabidopsis endomembranes, suppresses PTI and contributesto disease symptoms and bacterial growth in planta (DebRoyet al., 2004; Nomura et al., 2006). HopM1 mediates theUPS-dependent degradation of a key component of the plantvesicle trafficking system (Nomura et al., 2006). (2) XopJfrom Xcv has been proposed to localize, at least in part, tothe Golgi apparatus (Bartetzko et al., 2009). However, therespective analyses were performed in N. benthamiana, whereXopJ induces necrosis at 3–4 dpi (Thieme et al., 2007), rais-ing the possibility that the occasional localization in punctatestructures might be caused by morphological changesinduced by ongoing cell death. Nevertheless, XopJ inhibitssecGFP secretion and suppresses PTI (Bartetzko et al., 2009).In contrast with XopB, however, HopM1 and XopJ do notappear to affect ETI.

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Fig. 7 XopB suppresses the AvrBsT-dependent hypersensitive response (HR) in Xanthomonas campestris pv. vesicatoria (Xcv)-infected pepper. (a) Leavesof pepper (Capsicum annuum) cv ECW plants were inoculated with Xcv strains 75-3 (wt) and 75-3DavrBsT (DavrBsT) carrying the empty vector (ev),pBRM:xopB expressing xopB from plac, or pBRM:xopBA313V. Photographs were taken at 4 d post-inoculation (dpi). (b) Expression of xopB and xopBA313V

in Xcv strain 75-3 (wt) and 75-3DavrBsT (DavrBsT). Bacteria were grown overnight in liquid nutrient yeast glycerol (NYG) medium and incubated in hrp

gene-inducing medium for 3.5 h. Similar amounts were analyzed by immunoblotting using a XopB-specific antibody. (c) Bacterial multiplication of thestrains described in (a) was monitored over a period of 4 d. Values represent the mean of three samples from three different plants. Error bars indicatestandard deviations. Asterisks indicate statistically significant differences when compared with growth of the wild-type strain (t-test, P < 0.05). The experi-ments were repeated three times with similar results.

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The next challenge is the identification of plant targets,especially of XopB. The putative XopB target appears to beconserved in different plant families as the effector has a

virulence activity in pepper, the natural host plant of Xcv,and also in the nonhost Brassicaceae A. thaliana, as demon-strated by our protoplast assays. That XopB homologs are

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Fig. 8 XopB localizes to the Golgi apparatus and cytoplasm. (a) Confocal laser scanning microscopy of Nicotiana benthamiana 24 h afterAgrobacterium-mediated co-delivery of T-DNAs encoding XopB::GFP, XopBA313V::GFP, XopBK455R::GFP and GFP with T-DNA encoding Golgi-mCherry.Bars, 20 lm. (b) Ultrastructural localization of GFP fusion proteins by transmission electron microscopy in N. benthamiana mesophyll cells 48 h afterAgrobacterium-mediated transfer of T-DNAs encoding XopB::GFP, XopBA313V-GFP and GFP. Tissue samples were cryo-substituted and incubated with apolyclonal GFP-specific antibody and a gold particle-conjugated secondary antibody. For clarity, gold particles are marked with red arrows. Bars, 200 nm.d, dictyosome.






found in a wide range of plant-pathogenic bacteria(Table 1), infecting various host plants, supports this hypoth-esis. Interestingly, a recent study has shown that XopBinhibits yeast growth (Salomon et al., 2011). The applicationof caffeine, which induces cell wall stress, strongly increasesthe negative effect of XopB on yeast (Salomon et al., 2011).In the light of our results and the fact that vesicle transportis important for yeast cell wall assembly, for example, duringbudding (Smits et al., 2001), we believe that XopB targets aconserved component of eukaryotic vesicle trafficking.

Acknowledgements

We thank A. Urban, B. Rosinsky, C. Kretschmer, M. Jordan, S.Jahn and N. Bauer for excellent technical assistance. We are grate-ful to C. Lorenz and H. Berndt for providing unpublishedmaterial. This work was funded by grants from the DeutscheForschungsgemeinschaft to U.B., D.B., J.L. and D.S. (SFB 648‘Molekulare Mechanismen der Informationsverarbeitung in Pflan-zen’) and from the Bundesministerium fur Bildung und Forschungto J.L. and D.S. (‘tools, targets & therapeutics – ProNet-T3’).

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(b)

Fig. 9 XopB inhibits plant cell vesicle trafficking. (a) Confocal laser scanning microscopy of Nicotiana benthamiana leaves 2 d afterAgrobacterium-mediated delivery of T-DNA encoding secGFP alone (w ⁄ o) or T-DNA encoding the indicated effector proteins fused to a c-Myc tag.XopJ::c-Myc and AtSYP121_Sp2 served as positive control and XopJC235A::c-Myc as negative control. As additional controls, secGFP-expressing leaveswere infiltrated with 0.001% dimethyl sulfoxide (DMSO; mock) or brefeldin A (BFA; 10 lg ml)1 in 0.001% DMSO) and analyzed 20 min later. Bars, 20lm. (b) Expression of secGFP and effector genes in N. benthamiana. Inoculation of N. benthamiana leaves was carried out as described in (a). Infected tis-sue was harvested at 2 d post-inoculation (dpi) and analyzed by immunoblotting using GFP- and c-Myc-specific antibodies. Experiments were repeatedtwice with similar results.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 XopR- and XopS-AvrBs3D2 fusion proteins are expressedin Xcv 85*DhpaB.

Fig. S2 Individual deletion of xopB and xopS has no influence onbacterial growth.

Fig. S3 XopB and XopS do not suppress mitogen-activated pro-tein kinase (MAPK) activation in Arabidopsis.

Fig. S4 XopBA313V is impaired in cell death induction.

Fig. S5 XopB is associated with the membrane fraction of plantcells.

Fig. S6 Expression of GFP and xopB-GFP fusions in Nicotianabenthamiana.






Fig. S7 Subcellular localization of XopG::GFP.

Fig. S8 Subcellular localization of XopG, AvrBsT, AvrRxv andXopJ is not affected by co-expression of XopB.

Table S1 Bacterial strains and plasmids used in this study

Table S2 Oligonucleotides used in this study

Methods S1 Immunoblot-based detection of mitogen-activatedprotein kinase (MAPK) activation and membrane fractionation.

Please note: Wiley-Blackwell are not responsible for the contentor functionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the New Phytologist Central Office.

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